Reagent-impregnated test strips are commercially-available for a variety of predetermined analytes, such as glucose. These test strips are accurate, economical and relatively easy-to-use by an individual at home. The availability of such test strips has played a major role in the decentralization of the diagnostic market and has played an especially critical role in the development of home blood glucose monitoring that enables better glycemic control for diabetic individuals. In addition, other analyte detection devices designed for home use and that utilize amperometric, electrochemical detection of the predetermined analyte also are available. However, two important limitations remain in currently available technologies for home monitoring of a predetermined analyte, including the sample volume and the length of time that the assay requires. Generally, a test sample volume of between 5 .mu.L and 20 .mu.L (microliters) is required to perform an assay. The test sample is obtained by a finger puncture that produces fresh capillary blood. This is a relatively painful procedure, and the discomfort involved affects the willingness of an individual to perform assays as often as medically useful. In addition, currently-available assays require between 30 seconds and two minutes to complete.
Investigators have therefore shown an intense interest in developing electrochemical sensors that can be miniaturized, and therefore either be implanted subcutaneously or require a much smaller sample volume. Numerous approaches have been tried, including amperometric sensors based upon fiber electrodes and potentiometric sensors manufactured using techniques established in the semi-conductor industry. In contrast, the present invention measures a conductivity change in a thin polymer film, and accordingly has solved many of the problems inherent in miniaturized electrochemical sensors. Most importantly, an electrochemical sensor of the present invention can be manufactured in a planar format, using semi-conductor technologies, to provide an economical, disposable sensing element. Also, the sensor can be made sufficiently small such that a sample volume of 1 .mu.L or less can be assayed. The sensitivity of the detection method also has solved the general oxygen limitation problem frequently observed in electrochemical sensors that utilize oxidase enzymes. Therefore, in accordance with an important feature of the present invention, an electrochemical sensor that can be miniaturized; that provides rapid and accurate assays; that overcomes oxygen limitation problems; that is free of interferences attributed to common constituents in the test sample; and that can be produced economically is achieved.
In general, a diagnostic device of the present invention comprises a reaction zone wherein the predetermined analyte of interest interacts with a suitable oxidase enzyme to generate, either directly or indirectly, a dopant compound. The dopant compound is capable of oxidizing a conducting polymer to change the conductivity of the polymer. In addition, the diagnostic device further comprises a detection zone including a film or layer of conducting polymer and a microelectrode assembly. The change in conductivity of the conducting polymer layer as a result of the dopant compound is detected or measured by the microelectrode assembly. The change in conductivity then is correlated to the amount of predetermined analyte in the test sample. As will be demonstrated more fully hereinafter, an economical and reproducible conductive sensor, useful in assaying for a predetermined analyte that responds to oxidase chemistry, has been provided. The conductive sensor utilizes the properties of conducting polymers, avoids oxygen limitation problems in the normal concentration ranges of the predetermined analyte; can be manufactured by well-known semiconductor processing techniques; and does not rely upon a chemical reaction occurring at the microelectrode assembly.
Accordingly, one important feature of the conductive sensor of the present invention is the conducting polymer included in the detection zone of the conductive sensor. Several organic conducting polymers, such as polyacetylene, polypyrrole and polythiophene, are known. The organic conducting polymers have several potential applications in the fields of batteries, display devices, corrosion prevention in metals and semiconductors and in microelectronic devices such as diodes, transistors, sensors, light emitting devices and energy conversion and storage elements. However, organic conducting polymers possess several limitations that have hindered the use of organic conducting polymers in conductive sensors. In general, a conducting polymer useful in the conductive sensor of the present invention should display a sufficiently high conductivity for detectable and accurate measurements, and should be capable of being processed, reproducibly, into thin, uniform films. Although several conducting polymers possess one of these two necessary properties, only a limited number of conducting polymers possess both necessary properties.
For example, polyacetylene acts as an insulator exhibiting conductivities in the range of 10.sup.-10 S/cm to 10.sup.-13 S/cm (Siemens per centimeter). A conductivity in this range corresponds to the conductivity of known insulators, such as glass and DNA. However, polyacetylene can be doped using a variety of oxidizing or reducing agents, such as antimony pentafluoride, the halogens, arsenic pentafluoride or aluminum chloride. By doping, polyacetylene is converted into a highly-conducting polymer exhibiting a conductivity of approximately 10.sup.3 S/cm, corresponding to the conductivity of metals such as bismuth.
However, polyacetylene suffers from the drawbacks of extreme instability in air and a sharp drop in conductivity when an alkyl or other substituent group is introduced into the polymer. Accordingly, the instability of polyacetylene in the presence of oxygen, and the inability of substituted polyacetylenes to maintain a high conductivity, generally makes polyacetylene unsuitable as the conducting polymer in a conductive analyte sensor. As will be discussed more fully hereinafter, conducting polymers including alkyl or other substituent groups usually possess physical and mechanical properties making the substituted conducting polymer more easily processible into conducting polymeric films than the corresponding unsubstituted conducting polymer. Therefore, in the manufacture of conductive sensors it is desirable to use an easy-to-process conducting polymer, such as a conducting polymer that is stable in air and possesses suitable mechanical and physical properties, like solubility in organic solvents.
Similarly, the conducting polymer polypyrrole exhibits conductivities ranging from about 1 S/cm to about 100 S/cm. Investigators again found that placing substituent groups on either the nitrogen atom or a carbon atom of the heteroaromatic pyrrole ring decreases the conductivity of polypyrrole. For example, an unsubstituted polypyrrole, incorporating the tetrafluoroborate anion as the dopant compound, exhibits a conductivity of 40 S/cm, whereas the N-methyl derivative, incorporating the same dopant compound, exhibits a conductivity of 10.sup.-3 S/cm; the three-methyl derivative of pyrrole exhibits a conductivity of 4 S/cm; the 3,4-dimethyl derivative, a conductivity of 10 S/cm; and the 3,4-diphenyl derivative, a conductivity of 10.sup.-3 S/cm. Accordingly, substituted polypyrroles, although often demonstrating good physical properties, may not demonstrate a sufficient conductivity for use as the conducting polymer in a conductive analyte sensor.
As illustrated by the large conductivity drop in polypyrroles having substituents positioned on the pyrrole ring, even substituents as small as a methyl group introduce steric interactions sufficient to essentially destroy the conductivity of the polymer. However, as will be discussed more fully hereinafter, if a conducting polymer has a suitable substituent group present on the monomer units, reproducible processing of the conducting polymer into a thin film of uniform thickness is facilitated. Therefore, it would be desirable to provide a conductive sensor including a conducting polymer that is easy to process and that also exhibits a sufficiently high conductivity for sensitive and accurate analyte determinations.
The inability to add a substituent to a polypyrrole without significantly reducing the conductivity of the polymer is critical from a perspective of polymer processing. Polypyrrole and presently-known polypyrrole derivatives are intractable polymers that are insoluble in common organic solvents. Therefore, a polypyrrole cannot be processed conveniently. However, if a polypyrrole can be substituted without adversely affecting the electrical properties of the polymer, a processible polypyrrole may be developed.
The synthesis and conductivities of polypyrrole and substituted polypyrroles have been investigated extensively. The general references cited below include the information discussed above and further general information concerning polypyrroles. The representative references discussing the polypyrroles include:
M. S. Wrighton, Science 231, 32 (1986);
A. F. Diaz et al., J. Electroanal. Chem. 133, 233 (1982);
M. V. Rosenthal et al., J. Electroanal. Chem. and Interfac. Chem. 1, 297 (1985);
G. Bidan et al., Synth Met. 15, 51 (1986);
E. M. Genies et al., Synth. Met. 10, 27 (1984/85); and
J. P. Travers et al., Mol. Cryst. Liq. Cryst. 118, 149 (1985).
Investigators also have found that steric interactions in substituted polythiophenes are somewhat less dominant than those observed in substituted polypyrroles because the predominant destabilizing interactions in substituted pyrroles involve the hydrogen atom of the pyrrole nitrogen. These steric interactions are absent in the polythiophenes, and therefore, electronic effects are more predominant in substituted polythiophenes.
Polythiophene also is a well-studied, stable conducting polymer. Polythiophene resembles polypyrrole in that polythiophene can be cyclized between its conducting, i.e. oxidized, state and its nonconducting, i.e. neutral, state without significant chemical decomposition of the polymer and without appreciable degradation of the physical properties of the polymer. Polythiophene, like polypyrrole, exhibits conductivity changes in response both to the amount of dopant compound and to the specific dopant compound, such as molecular iodine, iridium chloride, arsenic(III) fluoride, phosphorus(V) fluoride, perchlorate, tetrafluoroborate, hexafluorophosphate, hydrogen sulfate, hexafluoroarsenate and trifluoromethylsulfonate.
Substituents placed on the heteroaromatic thiophene ring can affect the resulting conducting polymer. However, in contrast to pyrrole, ring substituents on thiophene do not seriously reduce the conductivity of the resulting heteroaromatic polymer. For example, it has been found that for 3-methylthiophene and 3,4-dimethylthiophene, the resulting substituted polythiophene exhibited an improved conductivity compared to the unsubstituted parent polythiophene, presumably due to enhanced order in the polymer chain of the substituted thiophene. Accordingly, unlike many pyrroles, substituents can be included on the thiophene monomer units to improve the processing properties of the resulting conducting polymer without adversely affecting the conductivity of the conducting polymer. Therefore, as a class, substituted polythiophenes are well-suited for use as the conducting polymers in a conductive analyte sensor.
The following are representative publications describing the synthesis and conductivity of polythiophene, substituted polythiophenes and the related poly(thienylene vinylenes):
G. Tourillon, "Handbook of Conducting Polymers," T. A. Skotheim, ed., Marcel Dekker, Inc., New York, 1986, p. 293;
R. J. Waltham et al., J. Phys. Chem. 87, 1459 (1983);
G. Tourillon et al., J. Polym. Sci. Polym. Phys. Ed. 22, 33 (1984);
G. Tourillon et al., J. Electroanal. Chem. 161, 51 (1984);
A. F. Diaz and J. Bargon, "Handbook of Conducting Polymers," T. A. Skotheim, ed., Marcel Dekker, Inc., New York 1986, p. 81;
G Tourillon et al., J. Phys. Chem. 87, 2289 (1983);
A. Czerwinski et al., J. Electrochem. Soc. 132, 2669 (1985);
K. Jen et al., J. Chem. Soc., Chem. Commun., 215 (1988); and
R. L. Elsenbaumer et al., Electronic Properties of Conjugated Polymers, 400 (1987).
From the studies on the polyacetylenes, polypyrroles and polythiophenes, and from related studies on other conducting polymers, it became apparent that a balance exists between the electronic effects and the steric effects introduced by the substituent on the monomer unit that renders a polymer of a substituted five or six member heteroaromatic ring more conducting or less conducting than the unsubstituted parent heteroaromatic compound. Therefore, it would be advantageous to utilize a conducting polymer having sufficient conductivity and suitable processing properties, such that the polymer can be used in a sensitive conductive analyte sensor of a diagnostic device to accurately determine the presence or concentration of a predetermined analyte in a liquid test sample.
Accordingly, the present invention is directed to a conductive sensor useful in a diagnostic assay for a predetermined analyte. More specifically, the conductive sensor is used in a diagnostic device to determine the presence or concentration of a predetermined analyte, like glucose, that is capable of interacting with an oxidase enzyme. The predetermined analyte and the oxidase interact in a reaction zone of the conductive sensor to generate, either directly or indirectly, a dopant compound. The dopant compound then migrates to a detection zone of the conductive sensor to oxidatively dope a layer or film of conducting polymer, thereby altering the conductivity of the layer of conducting polymer. The change in conductivity of the conducting polymer then is detected or measured by a microelectrode assembly, and can be correlated to the concentration of the predetermined analyte in the test sample.
The prior art includes teachings related to diagnostic assays using conducting polymers. However, the known prior art does not include any references suggesting or anticipating the miniaturized conductive sensor of the present invention, or its method of use. Furthermore, although several references disclose the use of conducting organic polymers in sensors, no known prior art reference discloses a sensor demonstrating the sensitivity and accuracy of the conductive sensor of the present invention. The prior art sensors are based upon a direct interaction of an analyte, usually a gas, with the conducting polymer. In contrast, the present conductive sensors utilize a response to a dopant compound generated, either directly or indirectly, by an interaction between the predetermined analyte and an oxidase enzyme. The dopant compound then migrates, or diffuses, into the layer of conducting polymer and alters the conductivity of the layer of conducting polymer, thereby allowing the determination of the presence or concentration of the predetermined analyte in the test sample.
The observed sensitivity of the conducting polymer to the concentration of the dopant compound is important in the development of sensors that are based upon oxidase enzymes. As will be demonstrated more fully hereinafter, a very small amount of the dopant compound generates a response. Therefore, only a small fraction of the available predetermined analyte in the test sample, such as less than 1% of the available analyte, is interacted and converted into the dopant molecule. As a result, the problem of an oxygen limitation, inherent in using oxidase enzymes, is overcome, and a sensitive determination of the total analyte concentration in the test sample is achieved.
The most common mode of interaction between an analyte and a conducting polymer is to affect the state of oxidation of the organic conducting polymer. As will be discussed more fully in the detailed description of the invention, the conductivity of the conducting polymer is related to the degree of oxidation of the conducting polymer, with the degree of oxidation, in turn related to the amount of dopant compound doping the conducting polymer. Therefore, the measured change in conductivity of the conducting polymer is correlated to the amount of dopant compound oxidizing the conducting polymer. In accordance with the method of the present invention, the amount of dopant compound in the layer of conducting polymer is directly related to the amount of predetermined analyte in the test sample. Consequently, a measurement of the rate of conductivity change of the layer of conducting polymer also is a measurement of the concentration of the predetermined analyte in the test sample.
For example, M. K. Malmros et al., in the publication, "A Semiconductive Polymer Film Sensor for Glucose", Biosensors 3, pp. 71-87 (1987/88), suggest using polyacetylene in a biosensor to quantitatively detect glucose. Malmros et al. teach the conversion of glucose by glucose oxidase to gluconic acid and hydrogen peroxide, and the subsequent conversion of iodide ion to molecular iodine, or triiodide anion, by the action of lactoperoxidase. Malmros et al. further teach that the iodine so generated can be used to change the conductivity of a polyacetylene film, particularly as a modifier of the effect of the peroxide.
Malmros et al. however do not teach a biosensor that solves the problems of assay interference and of oxygen limitation due to the low oxygen concentrations in biological samples. Malmros et al. teach the physical effect of the enzymatically generated iodine doping the polyacetylene polymer. The iodine is generated in solution through the addition of solution phase enzyme. Malmros et al. do not teach the incorporation of the enzymes or the iodide ion into solid phase films that can be laminated onto a conducting polymer film. Similarly, Malmros et al. do not teach a means of metering the test sample through a diffusion barrier or measuring the early kinetics of the reaction.
Moreover, the method disclosed by Malmros et al. cannot be employed in a biosensor due to the inherent limitations of the polyacetylene film. Most importantly, a polyacetylene cannot be processed using either solution casting or low temperature melt processes. As a result, thin polyacetylene films, such as 200 .ANG. in thickness, cannot be produced. A thick film of conducting polymer greatly reduces the sensitivity of the film to a dopant compound by requiring more dopant compound to achieve the same level of doping. This in turn increases the amount of predetermined analyte in the sample that must be converted into iodine to achieve a response. As will be demonstrated more fully hereinafter, the ability to cast thin films of the conducting polymer is an important aspect in providing reliable and accurate assay results.
Similarly, Malmros et al., in European Patent Application Publication No. 096,095, disclose an immunoassay utilizing doped conducting polymers, wherein the resistance of the polymer varies in response to the analyte in solution. Although Malmros et al. utilize a polyacetylene conducting polymer in an immunosensor, Malmros et al. do not teach or suggest assaying for a predetermined analyte capable of interacting with an oxidase enzyme to, either directly or indirectly, produce a dopant compound such that the rate of the conductivity change of the conducting polymer as the dopant compound oxidizes the conducting polymer is correlated to the concentration of the predetermined analyte in the test sample.
Wrighton et al., in European Patent Application Publication No. 185,941, disclose the use of conducting organic polymers as the active species in a chemical sensor. Wrighton et al. generally teach using the changes in physical properties of the conducting polymer as the active transduction into electrical signals. Specific examples cited in the patent include detection of oxygen gas, hydrogen gas, pH and enzyme substrate concentrations like glucose. The principal transduction mechanism described by Wrighton et al. is the direct use of the change in polymer conductivity induced by oxidation or by reduction. In contrast to the present invention, Wrighton et al. include the reaction catalyst, like an enzyme, in the conducting polymer matrix. Accordingly, the interaction occurs within the conducting polymer matrix. In the device and method of the present invention, the entire analyte-oxidase interaction occurs essentially in a reaction zone of the device to generate the dopant compound; the dopant compound then migrates from the reaction zone to a detection zone that includes the layer of conducting polymer. The rate of change of conductivity of the conducting polymer layer as the dopant compound diffuses from the reaction zone to the detection zone is used to measure the concentration of the predetermined analyte in the test sample. Wrighton et al. do not teach a method of integrating the glucose oxidation by oxygen into the oxidation properties of the polymer, nor do Wrighton et al. teach the conversion of glucose to iodine through coupled enzymatic reactions. Furthermore, Wrighton et al. do not teach the use of solution processible polymers; all of the polymers used by Wrighton et al. are grown electrochemically.
Wrighton et al., in U.S. Pat. No. 4,717,673, disclose polymer-based microsensors produced by anodic deposition of a conducting polymer onto a gold or platinum electrode surface. Wrighton et al. in U.S. Pat. No. 4,721,601 disclose microelectronic devices having electrodes functionalized with conducting polymers having specific properties. The devices disclosed in each patent measure resistance changes of electrochemically-grown polymers on electrode arrays having intergap spacings of less than two microns.
Elsenbaumer et al., in the publication "Processible, Environmentally Stable, Highly Conductive Forms of Polythiophene", Synth. Met., 18, pp. 277-282 (1987), describe a series of conducting poly(3-alkylthiophene) polymers having sufficient conductivity, stability, mechanical properties and processibility for a variety of applications. Jen et al., in the publication "Processible and Environmentally Stable Conducting Polymers", Polymeric Material, Vol. 13, pp. 79-84 (1985) also describe polythiophenes having good conductivity and mechanical properties.
Hotta et al., in "Novel Organosynthetic Routes to Polythiophene and Its Derivatives", Synth. Met., 26, pp. 267-279 (1988) teach the synthesis of poly(3-alkylthiophene) polymers having long alkyl side chains. Such conducting polymers are soluble in common organic solvents, are processible into uniform films and, when doped, exhibit excellent conductivity. Yoshino et al. in the three publications:
"Preparation and Properties of Conducting Heterocyclic Polymer Films by Chemical Method", Jpn. Journ. Appl. Phys., 23, pp. 2899-2900 (1984);
"Electrical and Optical Properties of Poly(3-alkylthiophene) in Liquid State", Solid State Commun., 67, pp. 1119-1121 (1988); and
"Absorption and Emission Spectral Changes in a Poly(3-alkylthiophene) Solution with Solvent and Temperature", Jpn. Journ. Appl. Phys., 26, pp. L2046-L2048 (1987), describe the synthesis and properties of the conducting poly(3-alkylthiophene) polymers. Jen et al., in U.S. Pat. No. 4,711,742, disclose doped and undoped conducting polymers that can be solubilized in organic solvents, with the resulting solution used to form conducting polymer films, including films of a poly(3-alkylthiophene).
Nagy et al., in the publication "Enzyme Electrode for Glucose Based on an Iodide Membrane Sensor," Analytica Chim. Acta., 66, pp. 443-455 (1973), describe the detection of glucose by potentiometrically monitoring the disappearance of iodide ion. Nagy et al. monitor the decrease in iodide activity at the electrode surface, whereas the present invention monitors the amount of a predetermined analyte in the test sample by measuring the rate of change of conductivity of the conducting polymer due to the generation of a dopant compound by an oxidase enzyme mediated interaction.
Mullen et al., in the publication, "Glucose Enzyme Electrode with Extended Linearity," Analytica. Chem. Acta., 183, pp. 59-66 (1986), describe a hydrogen peroxide-detecting electrode to assay whole blood for glucose. Mullen et al. disclose positioning a silane-treated membrane over a reactive enzyme layer to remove interferents and to extend the linearity of the electrode response to glucose concentration in undiluted whole blood. Similarly, Vadgama, in European Patent Application Publication No. 204,468, discloses a membrane for an enzyme-based electrode sensor to increase the range of linearity of the sensor response to generated hydrogen peroxide.
Other references relating to membranes, in general, or the detection of glucose in a test sample in particular, include M. B. McDonell and P. M. Vadgama, "Membranes: Separation Principles and Sensing", Selective Electrode Rev., 11, pp. 17-67 (1989); L. C. Clark et al., "Long-lived Implanted Silastic Drum Glucose Sensors", Trans. Am. Soc. Artif. Intern. Organs, Vol. XXXIV, pp. 323-328 (1987); P. Vadgama et al., "The Glucose Enzyme Electrode: Is Simple Peroxide Detection at a Needle Sensor Acceptable?", in Implantable Glucose Sensors - The State of the Art, International Symposium, Reisensburg, pp. 20-22 (1987); P. Vadgama, "Diffusion Limited Enzyme Electrodes", Anal. Uses of Immobilized Biological Compounds for Detection, Medical and Industrial Uses, pp. 359-377 (1988); and W. H. Mullen et al., "Design of Enzyme Electrodes for Measurements in diluted Blood", Analytical Proceedings, 24, pp. 147-148 (1987). These references also describe improved methods of detecting hydrogen peroxide in the assay of blood for glucose.
In addition to the above, the following references are representative of the state of the art of electromechanical sensors using heteroaromatic polymers:
Y. Ikariyama et al., Anal. Chem., 58, 1803 (1986);
C. Nylander et al., Anal. Chem. Symp. Ser., 17, (Chem Sens) 159 (1983);
H. S. White et al., J. Am Chem. Soc., 106, 5317 (1984);
G. P. Kittlesen et al., J. Am. Chem. Soc., 106, 7389 (1984);
Malmros, U.S. Pat. No. 4,444,892, disclosing a device having an analyte specific binding substance immobilized onto a semiconductive polymer to allow detection of a specific analyte;
European Patent Application Publication No. 193,154, disclosing immunosensors comprising a polypyrrole or polythiophene film having an antigen or antibody bound thereto; and
M. Umana and J. Waller, Anal. Chem., 58, 2979 (1986) disclose the occlusion, or trapping, of an enzyme, glucose oxidase, by electropolymerizing pyrrole in the presence of the enzyme. The polypyrrole containing the occluded enzyme then can be used to detect glucose. However, the method of the present invention differs in two critical respects. First, the detection mechanism in the present invention detects a generated oxidative dopant. The polymer film initially is present in its reduced, nonconducting form, and becomes conductive only as the dopant compound is produced enzymatically. In the publication of Umana and Waller, the polymer film initially is conductive and that conductivity is modulated by the enzymatic activity of the enzyme, that is serving as a dopant, and also by the generated peroxide. The second critical difference is that, in the present invention, the oxidase enzyme is retained in a distinct reaction zone layer that is in contact with the conducting polymer film in a detection zone layer.
Investigators also have studied various other problems associated with electrochemical sensors. For example, one major problem encountered in assays based on oxidase chemistry is the limited amount of molecular oxygen present in the system. In an oxidase catalyzed reaction, the predetermined analyte reacts with an equimolar amount of molecular oxygen. When the supply of molecular oxygen is depleted, the reaction ceases regardless of the presence of the oxidase enzyme and unreacted analyte. If no further molecular oxygen can enter the system, an erroneously low assay for the predetermined analyte results. If molecular oxygen can diffuse into the system, the oxidase-catalyzed reaction will continue, although slowly, until all the analyte is consumed. In this case, an accurate analyte is achieved, but the time needed to achieve the accurate assay is impractically long.
Therefore, investigators have sought methods to overcome the problem of oxygen limitation. One method is to mediate the oxidase-catalyzed reaction with a species other than oxygen. In this method, a compound such as ferrocene, a ferrocene derivative, ferricyanide couples or tetrathiafulvalene/tetracyanoquinone is used as a replacement for molecular oxygen. These compounds perform in a manner similar to molecular oxygen and are included in a sufficient amount such that all of the predetermined analyte in the test sample is oxidized. This method was used in an amperometric probe and is described by Cass et al. in Anal. Chem., 56, p. 607 (1984) and in Biosensors, Instrumentation and Processing, The World Biotech. Report, Vol. 1, Part 3, p. 125 (1987).
Another disclosed method of avoiding the oxygen limitation problem is to eliminate oxygen and oxygen substitutes altogether, and allow a direct electron transfer from the enzyme to the electrode. This method is disclosed by Y. Degani and A. Heller, J. Phys. Chem., 91, 1285 (1987). Furthermore, Vadgama, in European Patent Application Publication No. 204,468, disclosed avoiding oxygen limitations by using a silane-treated membrane to restrict entry of the predetermined analyte into the reaction enzyme layer. In contrast, the device and method of the present invention avoid the oxygen limitation problem kinetically, that is by detecting and measuring the concentration of the predetermined analyte before oxygen limitations occur and by limiting the amount of the predetermined analyte that contacts the reaction zone including the oxidase enzyme and the limited amount of molecular oxygen.
Another problem encountered in the design of a conductive sensor is the effect of interfering compounds that often are present in a test sample, such as the presence of ascorbate ion in the assay of a biological fluid for glucose. Investigators have found that in amperometric probes, interference from relatively easily oxidized compounds, such as ascorbate ion, phenolics, uric acid, acetaminophen and salicylates, occurs because the interfering compound is oxidized at the anode. Investigators accordingly have attempted to eliminate the affect of these interfering compounds. A common technique is exemplified in the publication of C. J. McNeil, et al., Biosensors, 3, p. 199-209 (1987/88), wherein the electroactive species was functionalized to lower its oxidation potential and thereby eliminate the interference in an immunosensor. I. Hannig et al., in "Improved Blood Compatibility at a Glucose Enzyme Electrode Used for Extra Corporeal Monitoring", Anal. Letters, 19(3&4), pp. 461-478 (1986), attempted to eliminate the effects of interferents by utilizing a thick enzyme layer to convert a relatively large amount of the available glucose. The corresponding large response for glucose conversion essentially swamped the interferent response.
In contrast, the method and device of the present invention utilizes a conductometric detection and measurement. Accordingly, an extremely low voltage can be used. The voltage is much lower than the oxidation potential of the interfering compounds, and therefore the interfering compounds are not oxidized. In addition, in the present invention, all chemical interactions occur in the reaction zone of the conductive sensor. The molecular iodine is generated in the reaction zone and migrates to dope the conducting polymer in the detection zone. Therefore, no direct interference is possible at the electrode.
However, the generated molecular iodine dopant compound is capable of interacting with various serum components, like ascorbate. If a sufficient amount of the molecular iodine interacts with serum components rather than doping the polymer, interferences are observed. Therefore, the method of the present invention relies upon fast assays to minimize interfering reactions of the generated molecular iodine. This is accomplished by the configuration of the sensor of the present invention, comprising a thin reaction zone and a thin detection zone, such that the molecular iodine is generated near the conducting polymer to quickly dope the conducting polymer before significant interfering reactions can occur. Furthermore, in the preferred embodiment of the present invention, it will be demonstrated that a semipermeable membrane utilized to meter the test sample into the reaction zone also selectively screens interfering compounds from the test sample, and therefore precluding an interaction with the generated molecular iodine.
Therefore, the method of the present invention allows the accurate assay of a predetermined analyte that is responsive to oxidase chemistry. The method utilizes a diagnostic device that includes a conductive sensor, wherein the conductive sensor comprises a reaction zone and a detection zone. The reaction zone of the conductive sensor is a thin film including the reagents necessary to interact with the predetermined analyte and to generate a dopant compound. The detection zone includes a film or layer of a conducting polymer and a microelectrode assembly such that the dopant compound can migrate to the detection zone to dope the conducting polymer, and such that the resulting change in conductivity, detected and measured by the microelectrode assembly, can be correlated to the amount of predetermined analyte in the test sample. The conductive sensor overcomes the disadvantages demonstrated by the prior art sensors, and therefore provides sensitive, accurate and reproducible assays; provides a fast assay, such as within 30 seconds, and preferably within 10 seconds, from a small blood sample, such as from about 0.1 .mu.l to about 5 .mu.l; eliminates the oxygen limitation problem associated with oxidase chemistry; eliminates the problems associated with interfering compounds present in the test sample; demonstrates excellent shelf stability; is economical and disposable; and is miniaturized and can be reproducibly manufactured by semiconductor processing techniques.